Investigating the role of the membrane in particulate methane monooxygenase (pMMO) structure and function

研究膜在颗粒甲烷单加氧酶 (pMMO) 结构和功能中的作用

• 批准号: 10676098
• 负责人: Frank Tucci
• 金额: $ 4.29万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676098
• 关键词: Active Sites; Address; Affect; Atmosphere; Bacteria; Bathing; Biochemical; Biological; Biophysics; Catalogs; Chemistry; Consumption; Copper; Cryoelectron Microscopy; Data; Detergents; Electron Transport; Environment; Environmental Health; Enzymes; Equation; Exhibits; Failure; Future; Goals; Health; Homeostasis; Human; Human Activities; Hydrophobicity; Interdisciplinary Study; Knowledge; Lipid Bilayers; Lipids; Liquid substance; Mass Spectrum Analysis; Membrane; Membrane Proteins; Metals; Methane; Methane Metabolism Pathway; Methane hydroxylase; Methanol; Methylococcaceae; Methylococcus capsulatus; Micelles; Microbe; Modeling; Molecular Structure; Natural Gas; Outcome; Oxidoreductase; Particulate; Pathway interactions; Physiological; Play; Process; Property; Proteins; Recovery; Reducing Agents; Research; Resolution; Risk; Role; Route; Scaffolding Protein; Scientist; Structure; System; Temperature; Testing; Training Programs; Transmembrane Domain; Visualization; anthropogenesis; atmospheric carbon dioxide; chemical reaction; climate change; climate instability; design; future pandemic; greenhouse gases; innovation; insight; interest; lipidomics; mimetics; nanodisk; oxidation; partial recovery; preservation; pressure; protein protein interaction; protein structure; reconstitution; tropospheric ozone

ABSTRACT The atmospheric content of greenhouse gases, such as methane, has long been ruled by microbes, such as methanotrophs. Recent human activity has upset this homeostasis, presenting an appreciable risk to human health in the present and future. Particulate methane monooxygenase (pMMO), a copper-dependent transmembrane enzyme from methanotrophic bacteria, oxidizes methane to methanol. Its ability to perform this difficult chemical reaction at ambient temperature and pressure offers a window into developing processes for conversion of biological natural gas to liquid (Bio-GTL) for climate change mitigation. Isolation of pMMO from the membranes and detergent solubilization have hindered past studies, resulting in a loss of enzymatic activity and distortion of protein structure. The failure of detergent micelles to recapitulate the physicochemical properties of the membrane may perturb functionally important metal centers, protein-lipid interactions, and protein-protein interactions. These challenges can be overcome by reconstituting pMMO in membrane mimetics like membrane scaffold protein (MSP) nanodiscs (NDs) and bicelles using homogeneous synthetic lipid bilayers, which enable partial recovery of pMMO activity and structure. The goal of this project is to explore the role of the native membrane in pMMO structure and function. Aim 1 is to optimize pMMO activity in detergent-free native ND systems. Preliminary data show that it is possible to reconstitute pMMO activity in NDs using native lipids extracted from methanotrophs. These native lipid NDs exhibit activity comparable to or better than pMMO in synthetic lipid NDs. Aim 2 is to characterize the membrane environment and its interaction with pMMO. This information will be used to optimize membrane mimetics for delineating the effects of lipid environment on pMMO structure and function. Untargeted and targeted lipidomics via mass spectrometry will be used to catalog the major lipid classes and identify specific lipid species, while also determining their relative abundances in native lipid extracts and membrane mimetics. Native mass spectrometry will provide insight into specific protein- lipid interactions that occur within membrane mimetics, informing the modeling of these interactions in cryoEM and crystal structures. Aim 3 is to characterize the structural effects of membrane mimetic environments on pMMO. More native-like membrane mimetics may allow for determination of a more biologically relevant pMMO structure by cryogenic electron microscopy (cryoEM). These studies will provide insight into the importance of the membrane for pMMO function, including crucial details about the pMMO structure, copper centers, transmembrane loops, protein-lipid interactions, protein-protein interactions, physiological reductant, active site, and mechanism. This project may also provide generalizable information about the importance of the native membrane environment for studying membrane proteins.

抽象的 大气中甲烷等温室气体的含量长期以来一直由微生物控制，例如 作为甲烷氧化菌。最近的人类活动扰乱了这种体内平衡，对人类造成了相当大的风险 现在和未来的健康。颗粒甲烷单加氧酶 (pMMO)，一种铜依赖性 来自甲烷氧化细菌的跨膜酶，将甲烷氧化为甲醇。它执行此操作的能力 在环境温度和压力下发生困难的化学反应为开发工艺提供了一个窗口 将生物天然气转化为液体（Bio-GTL）以缓解气候变化。从 pMMO 中分离 膜和去垢剂溶解阻碍了过去的研究，导致酶活性丧失和 蛋白质结构的扭曲。洗涤剂胶束未能重现洗涤剂的理化性质 该膜可能会扰乱功能上重要的金属中心、蛋白质-脂质相互作用以及蛋白质-蛋白质 互动。这些挑战可以通过在膜模拟物（如膜）中重建 pMMO 来克服 使用均质合成脂质双层的支架蛋白 (MSP) 纳米圆盘 (ND) 和 bicelles，使得 pMMO 活性和结构部分恢复。该项目的目标是探索本地人的作用 pMMO 膜的结构和功能。目标 1 是优化无去垢剂天然 ND 中的 pMMO 活性 系统。初步数据表明，可以使用天然脂质重建 ND 中的 pMMO 活性 从甲烷氧化菌中提取。这些天然脂质 ND 在以下方面表现出与 pMMO 相当或更好的活性 合成脂质 ND。目标 2 是表征膜环境及其与 pMMO 的相互作用。 该信息将用于优化膜模拟物，以描述脂质环境对 pMMO 结构和功能。通过质谱分析的非靶向和靶向脂质组学将用于编目 主要脂质类别并识别特定脂质种类，同时还确定它们在 天然脂质提取物和膜模拟物。天然质谱分析将提供对特定蛋白质的深入了解 膜模拟物内发生的脂质相互作用，为冷冻电镜中这些相互作用的建模提供信息 和晶体结构。目标 3 是表征膜模拟环境的结构效应 在 pMMO 上。更接近天然的膜模拟物可能有助于确定更具生物学相关性的膜模拟物 通过低温电子显微镜 (cryoEM) 观察 pMMO 结构。这些研究将提供深入了解 膜对于 pMMO 功能的重要性，包括有关 pMMO 结构、铜的关键细节 中心、跨膜环、蛋白质-脂质相互作用、蛋白质-蛋白质相互作用、生理还原剂、 活性位点和机制。该项目还可以提供有关该项目重要性的通用信息。 用于研究膜蛋白的天然膜环境。